[Construction and expression of recombinant baculovirus with Schistosoma japonicum SjPP gene].

Objective: To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells.

Methods: The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid.

Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus.

Background: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome.

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge.

Expression of open reading frames in silkworm pupal cDNA library.

A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open reading frame (ORF) sequences in Escherichia coli.

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori).

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition.

Oral administration activity determination of recombinant osteoprotegerin from silkworm larvae.

Osteoprotegerin (OPG) regulates the formation of osteoclasts and is involved in the regulation of bone resorption and remodeling. To investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant osteoprotegerin as a oral administration drug, the rh-OPG was expressed in the larvae of silkworm through the silkworm baculovirus expression system, and was orally administered to mice. Compared with the control, oral administration of rh-OPG was effective to decrease serum calcium concentration in normal mice, and block the bone loss induced by the loss of estrogen in ovariectomized mice.

Recombinant functional human lactoferrin expressed in baculovirus system.

Human lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. In this study, we amplified hLf cDNA by reverse transcription-polymerase chain reaction from normal human mammary gland. The nucleotide sequence of the hLf was identical to the known hLf.

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest.

The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex.

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable.

High level expression of functionally active human lactoferrin in silkworm larvae.

In this paper, recombinant human lactoferrin (rhLf) was expressed very well using Bombyx mori nuclear polyhedrosis baculovirus expression system. Infection of silkworm larvae with recombinant virus, vBm-hLf, the rhLf was efficiently secreted into larvae hemolymph and the concentration of product purified was about 65 microg/ml. The isolated rhLf molecular mass was approximately 78 kDa, lower than that of the human lactoferrin (hLf) standards, which may be due to incomplete glycosylation or protein degradation.

[Expression of phytase gene in Bombyx mori].

Phytase gene of Aspergillus niger 963 was cloned into baculovirus transfer vector. DNA of the recombinant vector was co-transfected with Bm-BacPAK6 DNA into BmN cells, and recombinant virus was selected by plaque assays. The recombinant virus was identified by Dot blot and Southern-blot with the specific probe for phytase gene.

[Research on TALF expression in Escherichia coli].

The expression of cDNA encoding Tachyleus auti-lipoposaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxin. First, the TALF gene was inserted into expression vectors pGEX-4T-2, pET22b and pET28a to construct recombinant expression plasmids. The recombinant plasmids were transformed to E.

Cloning and identification of methionine synthase gene from Pichia pastoris.

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively.

Expression of an antimicrobial peptide identified in the male reproductive system of rats.

Bin1b is a beta-defensins-like molecule originally isolated from the rat epididymis. Owing to its bactericidal activity, Bin1b may have therapeutic properties suitable for the treatment of sexually transmitted diseases. The amino terminus of the mature Bin1b peptide contains a conserved myristoylated Gly residue.

Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system.

Production of recombinant human osteoprotegrin from Trichoplusia ni cells and Bombyx mori larvae.

Human osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant OPG-Fc were constructed and successfully expressed in Trichoplusia ni cells and Bombyx mori larvae. Native SDS-PAGE and Western blot analysis revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori larvae compared with OPG and OPG-280.

[Expression of a human ribonuclease inhibitor variant in Escherichia coli and silkworm insect cell (Bombyx mori)].

Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR).

[Experimental research on spine fusion induced by tissue engineered bone].

Objective: To construct a reasonable substitute for the autograft bone in vitro and transplant it back into the rabbit models to induce the spine fusion.

Methods: The bone marrow stem cell from the seven New Zealand rabbits were cultured. Recombinant human bone morphogenetic protein-4 (rhBMP-4) that has been proved to be bioactive was obtained by the way of genetic engineering.

Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli.

The processing of human mitochondrial leucyl-tRNA synthetase had been previously investigated in insect cell. In the present work, the gene encoding human mitochondrial leucyl-tRNA synthetase with the same N-terminus as that processed in the mitochondria of insect cell was cloned and expressed in Escherichia coli. The enzyme was purified by affinity chromatography on Ni-NTA column.

[Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain)].

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain).

cDNA cloning and characterization of Agkistin, a new metalloproteinase from Agkistrodon halys.

Agkistin was a new snake venom metalloproteinase (SVMP) gene which was cloned from Agkistrodon halys. Its deduced amino acid sequence has two additional cysteines (Cys407 and Cys426) in the disintegrin domain compared to other RGD containing SVMPs. The full-length gene (Agkistin) and its disintegrin region (named Agkistin-s) were expressed by baculovirus expression system (pFastBac-Htb vector) with His-tag, and their platelet aggregation-inhibition activity was evaluated.

The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells.

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E.

Promoter activities in the baculovirus envelope glycoprotein gp64 gene.

Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites.

Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells.

BmK ITa1 cDNA was cloned and highly expressed in E. coli and insect cell. SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively.

[Identification of functional region of helicase gene promoter in Bombyx mori nuclear polyhedrosis virus].

DNA helicases are essential for replication of baculoviruses. It was found that the helicase gene promoter of Bombyx mori nuclear polyhedrosis virus, including 510 bp upstream of ATG, had both early and late RNA initiation sites and could be recognized by cellular RNA polymerase. Transient expression assays in uninfected Sf-21 cells indicated that the helicase gene promoter could be classified as a delayed-early gene promoter.